Semi-allogenic anti-tumour vaccine with HLA haplo-identical antigen-presenting cells

ABSTRACT

The present invention relates to semi-allogeneic antigen-presenting cells into which proteins and/or peptides or RNA or DNA or cDNA, respectively, encoding said proteins and/or peptides which are overexpressed in tumor cells or which are derived from autologous tumor cells or different tumor cells or different tumor cell lines have been introduced. Furthermore the invention relates to methods for the generation of these semi-allogeneic antigen-presenting cells as well as to the use thereof in the treatment of tumor diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP02/02595 filed Mar. 8, 2002, which claims priority under 35U.S.C. 119 to German Patent Application No. 101 12 851.7 filed Mar. 16,2001, the disclosures of which are all herein incorporated by referencein their entirety and this application is also a continuation of UnitedStates patent application No.: To be assigned, entitled SEMI-ALLOGENICANTI-TUMOUR VACCINE WITH HLA HAPLO-IDENTICAL ANTIGEN-PRESENTING CELLS(Inventors: Dolores SCHENDEL and Rudolf WANK) filed Sept. 15, 2003,which is a continuation of International Patent ApplicationPCT/EP02/02595 filed Mar. 8, 2002, which claims priority under 35 U.S.C.119 to German Patent Application No. 101 12 851.7 filed Mar. 16, 2001,the disclosures of which are all herein incorporated by reference intheir entirety.

The present invention relates to semi-allogeneic antigen-presentingcells into which proteins and/or peptides or RNA or DNA or cDNA encodingsaid proteins and/or peptides which are overexpressed in tumor cells orwhich are derived from tumor cells have been introduced. Furthermore theinvention relates to methods for the generation of these semi-allogeneicantigen-presenting cells as well as to the use thereof in the treatmentof tumor diseases.

Three recent developments suggest that cell-mediated immune reactionsmay result in a significant reduction in tumor load and in a similarlysignificant prolongation of the survival time of the patients in thecase of tumor diseases such as for example a metastatic renal cellcarcinoma (mRCC). Clinical data have already demonstrated a success inthe case of mRCC but it is also contemplated to transfer the sameprinciples to other tumors. In the following, three developments will bedescribed which form the basis of the approach according to theinvention.

I. Allogeneic Bone Marrow Transplantation for the Treatment ofMetastatic Renal Cell Carcinoma

Renal cell carcinoma patients subjected to allogeneic bone marrowtransplantation may achieve a complete or partial remission in 53% ofthe cases (1). The life expectancy may be significantly increased fromseveral months to several years. While important tumor loads may beeliminated, 53% of the patients develop a graft-versus-host disease(GVDH) accompanied by a GVDH-associated mortality of 5%.

The mechanism of tumor regression presumably is comparable to thatassociated with the elimination of hematological malignomas followingallogeneic bone marrow transplantation (BMT) (2). Activated Tlymphocytes recognize ligands consisting of peptides derived from aprotein expressed in tumor cells and MHC molecules (both of the class Iand the class II type). The MHC proteins are classified into threegroups two of which, namely the MHC class I and class II proteins, areof particular importance in the present invention. In humans the MHCclass I proteins are referred to as HLA-A, -B, -C. They are expressed onthe surface of almost any body cell and represent strong transplantationantigens showing a high degree of polymorphism. The MHC class IIproteins in humans are HLA-DP, -DQ, -DR and are for example expressed ondendritic cells and stimulated macrophages and also represent highlypolymorphic transplantation antigens.

Ligand recognition activates effector functions (for example cytokinesecretion and cytotoxicity) in various T cells whereby a destruction oftumor cells is effected. The parallel activation of T cells whichrecognize ligands composed of MHC and non-tumor associated peptides(i.e. alloreactions) may contribute to an optimal immune reaction by thesecretion of inflammatory cytokines which themselves promote theanti-tumor reaction. However, the reactions of these cells also play arole in GVHD.

II. Hybrid Vaccines Using Autologous Tumor Cells and AllogeneicDendritic Cells (DC)

Kugler et al. (3, 4) have shown that mRCC patients may be “vaccinated”by hybrid vaccines consisting of fused allogeneic dendritic cells andautologous tumor cells of non-related individuals. Following the“vaccination” the patients show a marked reduction in tumor load. Fewerpatients achieve a complete remission as compared to approach I, and anincreased life expectancy has not been observed so far. In contrast tothe treatment in approach I, however, the hybrid vaccine approach showsno or only a slight toxicity. After the termination of the vaccinationsome tumor patients suffered from a relapse presumably due to a lack ofsustenance of long-term T cell reactions. It may be possible to overcomethis problem by a continuous vaccination, however, the vaccination islimited by the availability of tumor cells serving as fusion partnersfor the allogeneic dendritic cells. Thus it is impossible to continuethe treatment if the tumor cells are declining.

III. RNA Obtained from Tumor Cells for the Generation of Vaccines on theBasis of Autologous DCs

Gilboa and coworkers (5-8) have developed a strategy for primingautologous DCs with autologous RNA prepared from tumor cells which canbe incorporated by DCs, translated into a protein and processed intopeptides for presentation with autologous MHC class I and class IImolecules. To provide an unlimited source of material for DC pulsing,RNA from tumor cells is transcribed back into cDNA which then serves asa template for in vitro transcription of further RNA. A PCRamplification enables the generation of material in unlimited amounts.The expression of RNA obtained from tumor cells in autologous DCsenables the expression on the surface of DCs of MHC-peptide ligandscorresponding to those expressed on the tumor cell surface. Theco-stimulatory molecules of the DC and the cytokines that it producesenable an optimal stimulation of T cell reactions. These vaccines showonly a slight toxicity, however, no remission comparable to those of Iand II has been published to date. This may be attributable to a lack inalloresponsive cells which are not activated in an autologous system.

In summary it may be concluded that the conventional therapy approachessuffer from substantial disadvantages preventing a long-term therapeuticsuccess or entraining severe undesirable effects.

Thus, as already described above, approach I suffers from the risk ofGVHD development occurring in 53% of cases and partially associated witha lethal outcome.

The approach described under II (Kugler et al.) has the disadvantagethat viable tumor cells of the patient are required to provide theMHC-peptide ligands and the approach thus is strongly limited in itsapplication period.

Although the therapy according to Gilboa and coworkers shows only aslight toxicity, no remarkable tumor remission has been published sofar.

Therefore it is an object of the present invention to provideantigen-presenting cells avoiding the disadvantages cited above whichthus are useful in the therapy of tumor diseases.

This object has been achieved by the features described in theindependent claims. Preferred embodiments are mentioned in the dependentclaims.

By using semi-allogeneic antigen-presenting cells into which have beenintroduced the proteins and/or peptides or RNA or DNA or cDNA,respectively, encoding said proteins and/or peptides which areoverexpressed in tumor cells or are derived from autologous tumor cellsor from several different tumor cell lines an immune reaction may beachieved which is advantageous for the treatment of tumor diseases.

Different from the approach of Kugler et al. the method according to theinvention for the generation of these semi-allogeneic antigen-presentingcells has the advantage that electrofusion is not necessary. Thefunction of the antigen-presenting cells thus is not disturbed byelectrofusion. An electrofusion can be avoided because transfection, forexample with RNA, utilizes the natural capability of antigen-presentingcells to incorporate such molecules.

The strategy according to the invention, i.e. the use of asemi-allogeneic vaccine provides a higher presentation efficiency of theligands consisting of MHC and tumor-associated peptide. Only about 10%of hybrid cells are generated by the approach of Kugler et al. whileaccording to the invention a clearly higher percentage ofantigen-presenting cells expressing the peptide ligand can be generated.

In this respect the approach used according to a preferred embodimentfor an (unlimited) generation of tumor-associated RNA is particularlyadvantageous by which e.g. RNA derived from autologous tumor cells isreverse transcribed, the cDNA is amplified by means of PCR andsubsequently the cDNA is transcribed into RNA.

This approach according to the invention has the advantage that alreadyminor amounts of tumor cells can provide sufficient RNA to form anunlimited source of cDNA for in vitro RNA transcription. By pulsing onthe basis of RNA it is thus possible to avoid the disadvantagesaccompanied by a limited amount of tumor cells. Long-term possibilitiesto perform vaccinations improve the reaction rates and the long-termprevalence thereof as compared to for example the approach according toKugler et al.

MHC molecules of semi-allogeneic antigen-presenting cells which do notcorrespond to the MHC molecules of the patients provide a stimulus foralloreactions. Thus, by using semi-allogeneic antigen-presenting cellsan enhanced immune reaction is provoked in comparison to the use ofautologous cells. Nevertheless the toxicity of the vaccine according tothe invention is much lower than that of the approaches described sofar.

The use of semi-allogeneic dendritic cells according to the inventionthus provides the necessary MHC matching according to the approach ofGilboa et al. while avoiding the disadvantage accompanying the use ofautologous dendritic cells that the function of autologous dendriticcells is weak and often associated with an advanced disease of cancerpatients.

According to the invention, antigen-presenting cells are prepared fromindividuals which are semi-allogeneic with respect to the patient. Thisis the case if the MHC molecules of antigen-presenting cells of arelated donor, e.g. father, mother, brothers and sisters, or offspring,are 50% identical to the MHC molecules of the patient. This may beschematically demonstrated as follows: if the patient HLA has thealleles a+c, close relatives being potential donors must have thefollowing allelic variants:

Parents: HLA ab or cd Brothers and sisters: HLA ad or bc Offspring: HLAae, af, ce, cf (mother = HLA ef)

Insofar, the semi-allogeneic antigen-presenting cells mentioned aboveare HLA-haploidentical with respect to those of the patient andtherefore may also be referred to as HLA-haploidenticalantigen-presenting cells.

HLA-haploidentical antigen-presenting cells have class I (HLA-A, -B, and-C) molecules in common with the patient which are encoded by the HLA-A,-B, and -C alleles of one chromosome. They also have class II molecules(HLA-DR, -DQ, and -DP) in common with the patient encoded by thecorresponding alleles of the same chromosome.

Following the isolation of the semi-allogeneic antigen-presenting cellsproteins and/or peptides or RNA or DNA or cDNA or cRNA, respectively,encoding said proteins and/or peptides derived from autologous tumorcells are introduced into these cells. In another embodiment proteinsand/or peptides or RNA, DNA, cDNA, or cRNA encoding said proteins and/orpeptides which are overexpressed in tumor cells are introduced. Theseare tumor-defined antigens, i.e. antigens specific for tumor cells andhaving a higher expression or markedly higher expression, respectively,in tumor cells as compared to normal cells. Preferably, suchtumor-defined antigens are selected from oncogenes, for exampleHER2/neu, proteins providing a growth advantage to the tumor and/orensuring its survival, e.g. PSMA, cell cycle regulatory proteins,transcription factors, e.g. WT-1, mucins, preferably MUC-1, proteinsinvolved in the regulation of cell division, preferably telomerase. Itshould be understood that the proteins, peptides, RNA, DNA, cDNA, andcRNA mentioned herein can also be prepared and used in a recombinantmanner.

As a result the antigen-presenting cells present a ligand consisting ofMHC and tumor-associated peptide which is recognized by T cells and thusinduces an immune reaction directed against the autologous tumor.

According to a preferred embodiment the use of antigen-presenting cellsof two different semi-allogeneic donors (i.e. for example mother andfather) is provided so that the MHCs presented by the semi-allogeneicDCs comprise all potential MHC class I and class II molecules of thepatient. The second chromosome of each donor additionally encodesallogeneic MHC molecules which may serve to induce alloreactions.

In order to provide MHC class I and class II peptide ligandscorresponding to those of the patient's tumor cells the already knownstrategy according to Gilboa and coworkers is used to provide anunlimited source of tumor-derived RNA for the pulsing ofantigen-presenting cells. An equivalent possibility is to pulse thesemi-allogeneic antigen-presenting cells with other sources of antigen,for example peptides and proteins (9).

According to one embodiment of the invention the proteins and/orpeptides or RNA or DNA, cDNA, or cRNA, respectively, encoding saidproteins and/or peptides overexpressed in tumor cells or derived fromautologous tumor cells are derived from tumor cells, preferablyautologous tumor cells or are overexpressed in said tumor cells,respectively, wherein the tumor cells comprise: cells of mesenchymaltumors, e.g. cells of sarcomas, carcinomas, preferably ovarian, mammaryand renal cell carcinomas, tumor cells of the hematopoietic system,preferably cells of leukemias and lymphomas, cells of epithelial tumors,cells of ectodermal tumors, e.g. melanomas, and cells of embryonictumors from undifferentiated tissue, preferably blastomas and teratomas.A particular example for the renal cell carcinomas which is examined inmore detail herein is the metastatic renal cell carcinoma. Thesemi-allogeneic antigen-presenting cells according to the invention maythen be used in the treatment of the tumors.

As already mentioned above the RNA from autologous tumor cells intendedfor the incorporation into the antigen-presenting cells may be reversetranscribed into cDNA for infinite amplification, the cDNA may beamplified by means of PCR, and the cDNA may be then transcribed intoRNA.

The administration of the antigen-presenting cells according to theinvention is carried out by the intravenous, subcutaneous orintramuscular routes wherein subcutaneous injection is preferred.

For the use in the present invention all types of antigen-presentingcells may be used. These particularly include monocytes/macrophages anddendritic cells of which the latter are preferred.

Another aspect of the present invention is that instead of components ofautologous tumor cells there is used a pool of components of severaldifferent tumor cell lines which is introduced into the semi-allogeneicantigen-presenting cells. These may then be used as vaccines in tumortreatment. To generate such a “generic” vaccine it is advantageous toprepare for example a pool of cDNA as described above of 3-5 differentRCCs which may then be used for all patients. Namely, it can be assumedthat most of the tumor-specific T cells primed against such pool-pulsedantigen-presenting cells can recognize autologous tumor cells due to thefact that they have peptide-MHC ligands in common. This means that it issufficient to generate a single product (i.e. a generic cDNA and thecorresponding cRNA transcribed in vitro) under GMP conditions (GMP=GoodManufacturing Practice). Afterwards it will be required to prepare onlythe semi-allogeneic dendritic cells for each patient who is to betreated by the vaccine therapy according to the invention under GMPconditions.

Eventually, the present invention comprises a pharmaceutical compositioncontaining semi-allogeneic antigen-presenting cells described abovewherein said composition preferably is in the form of a vaccine.

The Figures show:

FIG. 1 the principle of the generation of an allogeneic tumor vaccineaccording to Kugler et al. wherein an autologous tumor cell and anallogeneic dendritic cell of non-related individuals are fused;

FIG. 2 the approach according to the invention wherein a semi-allogeneicvaccine is generated by using HLA-haploidentical antigen-presentingcells.

The haplospecific vaccines present the following features:

-   -   ligands consisting of MHC and TAA (tumor associated antigen)        peptide for half of the patient alleles;    -   all alleles may be matched by means of two haploidentical DC        preparations;    -   HLA mismatched MHC-peptide ligands for stimulation of        alloreactions and T helper cells;    -   costimulatory molecules and cytokines derived from DCs for an        optimal promotion of the immune reactions (represented as “Y” in        the Figure).

In the following the basic principles of the invention as well as testmodels serving for the illustration thereof will be described in moredetail using dendritic cells as an example.

Two model systems will be used to establish the principles of thisapproach. The first uses a well characterized set of reagents from amelanoma model and the second uses a set of reagents from a renal cellcarcinoma model. The melanoma model has the advantage that the epitope(MHC-peptide ligand) seen by the T cell receptor (TCR) of the test clone(Tyr-F8) has been defined; this allows the efficiency of antigenpresentation by antigen-presenting cells such as dendritic cells to beanalyzed in a more quantitative fashion. The epitope for one renal cellcarcinoma specific T cell clone (TIL-26) has not yet been identified butthese cytotoxic T lymphocytes (CTLs) utilize highly characteristic TCRswhich can be identified and quantified in vitro with the method of realtime RT-PCR based on the molecular sequence of the TCR CDR3 region. Thisenables the assessment of the functional priming capacity of thedendritic cells by identifying T cells through these characteristic TCRsequences. Combined, these two models can be used to demonstrate thespecificity and the efficiency of this system for presentingtumor-associated peptides at the surface of the DCs and for activatingspecific T cell responses in vitro. These are essential experiments toestablish proof of principle prior to development of a phase I/IIclinical trial.

Melanoma System

The reagents for this model system were obtained from Prof. PeterSchrier of the University of Leiden. An HLA-A*O201-restricted CTL clone(Tyr F8) was isolated from a patient with advanced melanoma. Anautologous melanoma tumor cell line from the patient was alsoestablished. The specific ligand, i.e. the peptide derived from atumor-associated protein is known; it is derived from tyrosinase, anenzyme that is expressed at normal levels in healthy melanocytes butwhich is overexpressed in the melanoma. The MHC presenting molecule wasidentified to be encoded by the HLA-A*O201 allele. The cDNA fortyrosinase is available; the exact nine amino acid peptide derived fromthe tyrosinase protein that is presented in the peptide binding grooveof the HLA-A2 molecule is known and the Tyr F8 T cell clone is available(10).

The general outline of the experiment is first to prepare RNA from themelanoma line and then to prepare cDNA by reverse transcription. ThiscDNA is then amplified and used as a template to prepare in vitrotranscribed RNA (cRNA). The cRNA is used to pulse HLA-A2-positivedendritic cells (11, 12). These dendritic cells will take up the CRNA,and then translate it into proteins, one protein of which should be thetyrosinase enzyme. The tyrosine protein is processed intracellularly inthe dendritic cells into peptide fragments and the specific nine aminoacid epitope is loaded onto newly synthesized HLA-A2 molecules in theendoplasmic reticulum of the dendritic cells. These MHC-peptidecomplexes are then transported to the plasma membrane of the dendriticcell where they can be recognized by T cells. Tyr F8 cells recognizingthe appropriate MHC-peptide complex on the DC surface respond bysecreting interferon-gamma and activating the cytolytic mechanism,allowing them to kill the dendritic cell. This activation can beassessed by ELISA detection of interferon-gamma secretion by the Tyr F8clone or by using a standard chromium release assay, labeling thedendritic cells as target cells and measuring their lysis in thepresence of Tyr F8 cells. The activation of the Tyr F8 cells is veryspecific so that MHC molecules carrying peptides derived from proteinsother then tyrosinase can not activate the Tyr F8 cells and a certainthreshold of these specific MHC-tyrosinase peptide complexes must bepresented by the DC in order to optimally stimulate the activity of theTyr F8 cells.

To determine whether the original cRNA can provide the necessarystarting material to allow the dendritic cell to fulfill all thesesteps, cRNA-pulsed dendritic cells are used as stimulating cells toactivate the Tyr F8 cells. Because the melanoma tumor cells yield avariety of different RNAs, transcripts for tyrosinase represent only onecomponent of a much larger pool. The capacity of this mixed cRNA pool toprovide the tyrosinase epitope can be compared to cRNA made from theisolated tyrosinase cDNA (i.e. only one RNA species). A relativeassessment of the level of HLA-A2-tyrosinase-peptide complexes can bedetermined by pulsing dendritic cells with varying amounts of test cRNAand comparing their stimulatory capacity with dendritic cells that havebeen pulsed with varying known amounts of synthetic peptide,representing the nine amino acid epitope of tyrosinase. The thresholdlevel at which the Tyr F8 cells can no longer detect the correctMHC-peptide complex can be determined by assessing the amount ofinterferon-gamma released by Tyr F8 cells using a specific ELISA assay.

Renal Cell Carcinoma System

These reagents have been established by the inventors. We have isolateda CTL line specific for renal cell carcinoma from a renal cell carcinomapatient and T cell clones have been established from this T cell line(TIL-26). Autologous tumor cells were also obtained from this patient(RCC-26). The MHC restriction molecule presenting a tumor-associatedpeptide displayed by RCC-26 cells has been identified as theHLA-A*O201-encoded molecule. When the TIL-26 cells were exposed to theautologous RCC-26 tumor cells they were activated to secreteinterferon-gamma and they were able to lyse the autologous RCC-26 tumorcells (13).

The general outline of the experiment is similar to that described forthe melanoma system. In the first step, RNA will be made from RCC-26cells and cDNA will be prepared by reverse transcription. This serves asthe template to generate cRNA by in vitro transcription. The cRNA isused to pulse the dendritic cells and these dendritic cells are assessedfor their capacity to activate the TIL-26 cells to secreteinterferon-gamma. The dendritic cells are also assessed as target cellsfollowing pulsing with cRNA derived from RCC-26 cells in cytotoxicityassays using activated TIL-26 cells. The efficiency of stimulation ofthe dendritic cells can be compared with that of the RCC-26 cells, inassays measuring interferon-gamma secretion by the TIL-26 cells and astarget cells in cytotoxicity assays.

These two sets of experiments clearly demonstrate that the procedure forgenerating MHC-peptide complexes using cRNA-pulsed allogeneic(HLA-A*O2O1) dendritic cells is functional.

Induction of T Cell Responses by cRNA-Pulsed Allogeneic Dendritic Cells:

To assess the capacity of the cRNA-pulsed dendritic cells to primeallogeneic T cells in vitro two experiments are performed in vitro.First, it was shown that naive T cells from donor DS (DS=anonymousdonor) can be primed in vitro with RCC-26 tumor cells to recognize anepitope that is apparently the same as that seen by TIL-26 (14). LikeTIL-26 cells, the T cells from donor DS express TCRα chains that arehighly homologous to those expressed by TIL-26 cells (15). The TCRsequence of these T cells can be quantified by isolating RNA from a testsample and using TCR-specific primers to analyze the amount oftranscripts in the samples using the method of real time RT-PCR (14).First, allogeneic HLA-A*O2O1-positive dendritic cells pulsed with cRNA(RCC-26-derived) are used as stimulating cells in mixed lymphocytecultures using peripheral blood lymphocytes (PBL) of donor DS asresponding cells. The emergence of the marker (Va2O) T cells recognizingthe same epitope seen by TIL-26 cells is monitored by looking forincreases in transcripts with characteristic Va2O sequences. Aquantitative assessment can be made using real time RT-PCR comparing thefrequency of such transcripts in the unstimulated PBLs and followingstimulation with the cRNA-pulsed dendritic cells in vitro after variousrounds of restimulation. Autologous dendritic cells derived from donorDS pulsed with cRNA (RCC-26-derived) are used as target cells andcompared directly to RCC-26 tumor cells using, as cytotoxic effectorcells, the PBLs primed with cRNA-pulsed dendritic cells versus PBLsprimed with intact RCC-26 tumor cells.

As a second test system, T cells from an HLA-A*O201 donor are stimulatedwith cRNA-pulsed (melanoma-derived) dendritic cells which are derivedfrom a donor expressing HLA-A*O201 in mixed lymphocyte-dendritic cellcultures. The development of T cells recognizing the tyrosine-specificligand is assessed by standard flow cytometry using HLA-A2-tyrosinasepeptide-specific tetramers (7).

Pulsing of Dendritic Cells with Mixed cRNA Samples:

It is currently assumend that immune responses generated againstmultiple epitopes will provide the best possibility to eliminate tumorcells and to protect against tumor relapse. This is based onobservations that CTLs primed in vivo against individual NHC-peptideligands have led to the generation of CTLs specific for the MHC-peptideligand and, in some instances, to tumor regression. But tumor variantsmay emerge that no longer express the MHC molecules or no longer expressthe protein from which the specific peptide is derived. If CTLs of manydifferent MHC-peptide ligand specificities are generated then it may beless feasible for tumor variants to emerge because individual tumorcells would need to express different mutations in all MHC molecules andproteins providing peptides, in order to escape detection by CTLs withmany different specificities.

Therefore, it ise optimal to pool cRNA from several tumor cell lines inorder to provide many different tumor-associated peptides for immuneattack. Similarly, it is also better to have several different MHCmolecules shared by the responding T cells and the stimulating dendriticcells. This is achieved according to the strategy described herein byusing HLA-haploidentical dendritic cells derived from family members ofthe donor of the T cells, as described below. However, it must beensured that by pooling cRNA from different tumors the required densityof MHC-peptide ligands characteristic for specific epitopes of one tumorcell line is still retained. This can be addressed experimentally bycombining cRNA from the melanoma line with cRNA from the RCC-26 line.This pooled cRNA is then used to pulse allogeneic (HLA-A*O2O1-positive)dendritic cells and to assess whether these dendritic cells are stillcapable of activating both the melanoma-specific CTL clone, namely TyrF8, and the RCC-specific clone, namely TIL-26. If this experiment showsthat pooled cRNA (mixed at a ratio of 1:1 from the two tumor cRNAs)functions to activate both T cell clones, then variations in the ratioof the two cRNAs can be used to determine the degree to which a singlecRNA sample can be diluted and still functions in DC processing andpresentation, respectively. On the basis of pooled cRNA from severaldifferent tumor lines (for example, three RCC lines) a vaccine will thenbe generated and additional cRNAs will be added to this pool which havebeen derived from known genes expressed in various renal cell carcinomasthat are also potential target molecules for specific CTLs. These couldinclude HER2/neu, MUC1, PSMA, WT-1, telomerase and several othercandidates (9). The influence of the addition of these individual cRNAspecies can be assessed by adding them to the cRNA of RCC-26 and testingthe pulsed dendritic cells for their ability to activate TIL-26 cells.

Comparison of Autologous Versus HLA-Haploidentical Dendritic Cells asAntigen-Presenting Cells:

The experiments described above were designed to establish the use ofcRNA-pulsed allogeneic dendritic cells obtained from unrelated donorsthat share only HLA-A*0201-encoded molecules with the responding Tcells. The next step is then to demonstrate that HLA-haploidenticaldendritic cells can prime T cells against tumor-associated epitopestransferred into the dendritic cells by cRNA. This strategy is chosenbecause HLA-haploidentical DCs share three class I alleles (HLA-A, B,and C) and three class II alleles (HLA-DR, -DQ and -DP) with the Tcells. The different class I molecules can each bind a unique set ofpeptides and prime different CD8+ CTLs, and the three class II-encodedmolecules also each bind different sets of peptides that are distinctfrom those of the class I molecules. These class II-peptide ligandsactivate another type of T lymphocyte, the CD4 cells, which can providecytokines that support the optimal development of an immune response orfunction directly as effector cells (11).

The experiments analyzing the role of HLA-haploidentical dendritic cellswill be done by comparing autologous dendritic cells prepared from anormal control donor (donor #1) and HLA-haploidentical dendritic cellsprepared from a selected HLA-typed family member (donor #2) (i.e. aparent or sibling is selected to have one HLA haplotype in common withdonor 1 and the second HLA-haplotype is mismatched; see FIG. 2). DCsprepared from both donors will be pulsed with the cRNA of RCC-26 or themelanoma line. The resulting DCs will first be tested for their capacityto stimulate interferon-gamma secretion from Tyr F8 and TIL-26 cells, asdescribed above. In the next step, PBLs from donor #1 will be stimulatedin vitro with cRNA-pulsed autologous dendritic cells or theHLA-haploidentical dendritic cells of donor 2. Following two or morerounds of restimulation, the presence of T cells recognizing epitopespresented by different MHC molecules (both class I and class II) will beassessed by measuring their interferon-gamma secretion followingstimulation with cRNA-pulsed autologous dendritic cells in the absenceand presence of specific monoclonal antibodies. The MHC class I-specificmonoclonal antibodies (W6/32) will block interactions of CD8+ CTLs withthe dendritic cells. Decreased interferon-gamma secretion in thepresence of monoclonal antibodies specific for HLA-A, -B or -C moleculeswill demonstrate the degree to which CTLs seeing their peptides inassociation with different class I molecules are present. The presenceof class II-restricted CD4+ cells can be elucidated by the same approachbut instead using class II-specific monoclonal antibodies, reacting withHLA-DR, -DQ and -DP molecules (17).

The generation of cRNA according to the invention will be illustrated bythe following flow chart:

Flow Chart for Preparation of cRNA

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1. A method for the generation of HLA-haploidentical antigen-presentingcells for the treatment of tumor diseases in a patient comprising thefollowing steps: providing a patient to be treated; providingantigen-presenting cells from a semi-allogeneic donor which areHLA-haploidentical with respect to those of the patient, whereinHLA-haploidentical antigen-presenting cells have class I and class IImolecules in common with the patient; and introducing proteins and/orpeptides or RNA or DNA or cDNA encoding said proteins and/or peptidesinto the HLA-haploidentical antigen-presenting cells, wherein saidproteins and/or peptides are tumor-defined antigens or wherein said RNAor DNA or cDNA encodes tumor-defined antigens, wherein the tumor-definedantigens are antigens overexpressed in the tumor cells, wherein thetumor-defined antigens are selected from the group consisting ofoncogenes, proteins providing a growth advantage to the tumor and/orensuring its survival, cell cycle regulatory proteins, transcriptionfactors, mucins, and proteins involved in the regulation of celldivision.
 2. A method for the generation of HLA-haploidentical antigenpresenting cells for the treatment of tumor diseases in a patientcomprising the following steps: providing a patient to be treated;providing antigen-presenting cells from a semi-allogeneic donor whichare HLA-haploidentical with respect to those of the patient, whereinHLA-haploidentical antigen-presenting cells have class I and class IImolecules in common with the patient, wherein antigen-presenting cellsof two different HLA-haploidentical individuals are used; andintroducing proteins and/or peptides or RNA or DNA or cDNA encoding saidproteins and/or peptides into the HLA-haploidentical antigen-presentingcells, wherein said proteins and/or peptides are tumor-defined antigensor wherein said RNA or DNA or cDNA encodes tumor-defined antigens,wherein the tumor-defined antigens are antigens overexpressed in thetumor cells, wherein the tumor-defined antigens are selected from thegroup consisting of oncogenes, proteins providing a growth advantage tothe tumor and/or ensuring its survival, cell cycle regulatory proteins,transcription factors, mucins, and proteins involved in the regulationof cell division.
 3. A method of treatment of tumor diseases in apatient comprising administering to said patient a therapeuticallyeffective amount of semi-allogeneic HLA-haploidentical antigenpresenting cells into which proteins and/or peptides or RNA or DNA orcDNA encoding said proteins and/or peptides have been introduced,wherein HLA-haploidentical antigen-presenting cells have class I andclass II molecules in common with the patient, and wherein said proteinsand/or peptides are overexpressed in tumor cells or derived fromautologous tumor cells, and wherein said HLA-haploidenticalantigen-presenting cells are administered by the intravenous,subcutaneous or intramuscular route.
 4. A method of treatment of tumordiseases in a patient comprising administering to said patient atherapeutically effective amount of semi-allogeneic HLA-haploidenticalantigen presenting cells into which proteins and/or peptides or RNA orDNA or cDNA encoding said proteins and/or peptides have been introduced,wherein HLA-haploidentical antigen-presenting cells have class I andclass II molecules in common with the patient, whereinHLA-haploidentical antigen-presenting cells of two differentHLA-haploidentical individuals are used; and wherein said proteinsand/or peptides are overexpressed in tumor cells or derived fromautologous tumor cells, and wherein said HLA-haploidenticalantigen-presenting cells are administered by the intravenous,subcutaneous or intramuscular route.